Project Summary/Abstract The goal of this program is to understand how informational molecules that pattern tissues and embryos distribute in space and time during development. We study signaling in Drosophila, investigating the Decapentaplegic, Hedgehog, FGF, EGF, Wg, Notch-Delta and Bicoid morphogen signaling proteins. Our thesis is that the mechanisms that move these proteins from their sources and distribute them to their targets involve cellular machines and organelles whose actions precisely regulate protein movement. Our work has identified novel processes that mediate movement of morphogen signaling proteins in tissues and embryos, and this proposal describes the approaches we will take to further characterize these processes and the machines and organelles that drive them. This work has its origins in two separate investigations. The first began with an analysis of the roles and functions of the engrailed (en) segmentation gene. Like most vertebrates, the Drosophila embryo undergoes a mid-blastula transition (MBT) prior to gastrulation, but the early stages of Drosophila development have unusual features - 13 synchronous, rapid nuclear divisions without cytokinesis. Although the dogma has been that the zygotic genome does not contribute to pre-MBT development, we discovered that zygotic gene expression in nuclear cycle 2 embryos is essential for normal development. We discovered functionally important zygotic en expression in nuclear cycle 2 embryos and identified a small cohort of genes expressed by the pre-blastoderm embryo. We also discovered that the Bicoid concentration gradient that organizes the embryo A/P axis forms from protein that is made in stage 14 oocytes and functions prior to nuclear cycle 7. These findings are the basis for the proposed program that investigates patterning in the early embryo and that already reveals that our understanding of this early, critical stage of development must change radically. The second began with our discovery of cytonemes, specialized filopodia that are involved in cell-cell signaling. This discovery led us to propose that signaling proteins move between cells in a manner similar to the way neurotransmitters exchange between pre- and post-synaptic cells ? by transferring between signaling cells at synapses. Our work has established that synapses are present in the Drosophila wing imaginal disc at sites of cytoneme contact, that they involve proteins that have previously been shown to function and to be required at neuronal synapses, and that they are essential for paracrine signaling between non-neuronal cells. We have also obtained strong experimental evidence that cytonemes ferry signaling proteins between producing and receiving cells and we have identified several unexpected properties of cytonemes that have significant implications for mechanisms of pathfinding and signal transduction. The work we pursue develops new tools for imaging cytonemes and builds upon our previous findings to determine the roles, composition and functions of these remarkable organelles and this fascinating mechanism of contact-based signaling.